Abstract
Quantum diamond sensors containing Nitrogen Vacancy (NV) centers were integrated into the culture of spontaneously electrically active cultures of live mouse primary cortical neurons. Two diamond formats were used enabling extracellular studies of cells cultured directly on a planar diamond plate and intracellular studies via endocytosis mediated integration of nanoscale diamond particles within cells. Over a 14 day culture period functional neuronal networks formed as confirmed by endogenous calcium transients detected via fluorescent imaging of a calcium probe (Fluo-5 AM). Spatially correlated images of photoluminescence from NV centers were also recorded demonstrating successful acquisition of optically detected magnetic resonance from regions of endogenously firing neuronal cultures. Further, a fast and simple measurement protocol based on acquisition of NV photoluminescence without and with the application of near resonant microwaves is presented. This protocol was applied to live signaling cells and dead cells. The relative photoluminescence from NVs with and without microwaves varied for the case of live as compared to dead cells warranting future investigation. Overall this work presents a protocol for integration of NV diamond sensors in a spontaneously active network of neuronal cells of relevance to the field of neuroscience. Photoluminescence from NV centers was detected with high spatiotemporal resolution using measurement protocols potentially capable of single shot detection of neuronal activity. It is anticipated that the methodologies introduced in this work will underpin the establishment of NV diamond sensors as a radically new measurement platform capable of rapid, non-destructive functional studies of cells.
Highlights
Spontaneous neuronal activity is a fundamental feature of all known neuronal networks and has been well-documented in such various systems as the cerebral cortex, hippocampus, retina, and spinal cord, both in vitro and in vivo [1,2,3]
Live images of cells obtained using differential interference contrast (DIC) microscopy are shown for the nanodiamond (Figure 3Aiii) and diamond plate protocols (Figure 3Biii) which display the morphology of cells and network structure consistent with that expected of cortical neuronal networks after 14 days of culture
The corresponding frequency domain results of data obtained for 5 s is shown Figures 4Aiv,Biv. For both diamond formats there is a dominant frequency of 0.5 Hz suggesting cultures integrated with diamond plates and nanodiamonds both support similar rates of activity consummate with slow rhythms seen in dissociated neuronal cultures [16]
Summary
Spontaneous neuronal activity is a fundamental feature of all known neuronal networks and has been well-documented in such various systems as the cerebral cortex, hippocampus, retina, and spinal cord, both in vitro and in vivo [1,2,3]. In dissociated primary cortical cultures, as with other neuronal networks, the development of spontaneous electrical activity undergoes a global increase in complexity over time, with neurons exhibiting network-wide activity in characteristic. A prominent feature of network maturation in vitro is the development of spontaneous synchronized bursts of activity across the network, which strongly correlate with synaptic density/strength [6, 7]. In vitro cultures of isolated primary cortical neurons represent a valuable biological system for neurobiologists to study the development of neuronal networks, their activity, and the role this activity has in functional maturation of cortical circuits [8]. Cortical neurons within culture self-organize to form spontaneously active networks generating action potentials and intracellular calcium transients that resemble many basic properties of spontaneous activity observed in the immature neocortex in vivo [3]
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